Whole Organ Blood and Lymphatic Vessels Imaging (WOBLI)

Thin section histology is limited in providing 3D structural information, particularly of the intricate morphology of the vasculature. Availability of high spatial resolution imaging for thick samples, would overcome the restriction dictated by low light penetration. Our study aimed at optimizing the procedure for efficient and affordable tissue clearing, along with an appropriate immunofluorescence labeling that will be applicable for high resolution imaging of blood and lymphatic vessels. The new procedure, termed whole organ blood and lymphatic vessels imaging (WOBLI), is based on two previously reported methods, CLARITY and ScaleA2. We used this procedure for the analysis of isolated whole ovary, uterus, lung and liver. These organs were subjected to passive clearing, following fixation, immunolabeling and embedding in hydrogel. Cleared specimens were immersed in ScaleA2 solution until transparency was achieved and imaged using light sheet microscopy. We demonstrate that WOBLI allows detailed analysis and generation of structural information of the lymphatic and blood vasculature from thick slices and more importantly, from whole organs. We conclude that WOBLI offers the advantages of morphology and fluorescence preservation with efficient clearing. Furthermore, WOBLI provides a robust, cost-effective method for generation of transparent specimens, allowing high resolution, 3D-imaging of blood and lymphatic vessels networks.

alexa fluor 488 (1: 250, Molecular Probes, USA) was used as secondary antibody. Next, brains were cleared as described in the materials and methods section. Experiments were carried out in accordance with the Israeli law, and were approved by the Israel Institute for Biological Research (IIBR) Institutional Animal Care and Use Committee.
Microscope was equipped with a single lens configuration -4X objective -LVBT 4X UM2-BG, with an adjustable refractive index collar set to the RI of 1.38. Samples were glued to the sample holder and placed in an imaging chamber made of 100% quartz (LaVision BioTec) filled with ScaleA2 solution and illuminated from the side by the laser light. Images were acquired by an Andor Neo sCMOS camera (2,560 × 2,160, pixel size 6.5 µm x 6.5 µm, Andor). Z stacks were acquired in 2 µm steps, larger fields of view were imaged by tiling with 10% overlap. Images were stitched with Arivis software.

Tissue clearing by CLARITY
Tissues were cleared according to the protocol specified by Tomer et. al. 2 . Briefly, 4 months old female Vecad cre /tdTomato flox/stop/flox transgenic mice were transcardially perfused with 20 ml of ice-cold PBS, followed by 20 ml HM solution (4% acrylamide, 0.05% bisacrylamide, 4% PFA, 0.25% (wt/vol) VA-044 thermal initiator, in 1% PBS) at an approximate rate of 10ml/min. organs were then placed in HM solution for 1 day at 4°C. Samples were then de gassed using nitrogen for 5-10 min and immediately incubated at 37°C for 3 hours or until polymerization. After gel formation, residual gel was removed and samples were immersed in clearing solution (200mM boric acid, 4% SDS) at 37°C for passive clearing. After complete clearing, samples were washed twice in PBST at room temperature, and placed in FocusClear (Celexplorer, Taiwan) for refractive index matching.
Detailed materials information is found in the main text.

Intensity profile analysis
Images of ovaries cleared by WOBLI, CLARITY, CLARITY with passive hydrogel immersion and ScaleA2 were taken with light sheet microscope. The distance from the lens at the X axis was kept constant. Two-dimensional slices from depths 100 µm, 0.5 mm and 1 mm were chosen for intensity profile analysis. A line was drawn using ImageJ software, and the intensity profile along this line was extracted. The line was always placed from the left tissue edge. If the line was bigger than the tissue seen in the image, it was placed so the tissue will be close to the center of the line. The same line was then placed on the background of the image (where no tissue is imaged) and the average of these values was subtracted from the previous intensity values to form a normalized intensity profile. The normalized intensity values are shown as a function of the distance in pixels on the drawn line.
Livers were cleared and imaged using a light-sheet fluorescent microscope. 3D projection of liver is presented in the movie Image dimensions: 1200µm × 600µm × 600µm XYZ.
Uteri were cleared and imaged using a light-sheet fluorescent microscope. 3D projection of uterus is presented in the movie Image dimensions: 1200µm × 1600µm × 400µm, XYZ.
Ovaries were cleared and imaged using a light-sheet fluorescent microscope. 3D projection of ovary is presented in the movie. Image dimensions: 3200µm × 1600µm ×